Separations of charged particles, in particular physical mixtures of chemical species, are important analytical operations. Relevant chemical species include non-biological charged species, such as synthetic polymers, and biological charged species, such as DNA, RNA, or proteins (A. J. Kostichka et al., 1992, Bio/Technology 10:78). Separations of mixtures of DNA fragments are particularly important.
For example, the Human Genome Project demonstrates the need for powerful DNA fragment separation methods and apparatus. This project is an ambitious, international effort to improve human genetic maps, to sequence fully the genomes of humans and several model organisms by 2006, and to develop computational tools for storing and accessing the burgeoning information. This project requires a technological infrastructure capable of supplying high-quality sequence information in a rapid and cost-effective manner.
To sequence fully the human genome, which has approximately 3.times.10.sup.9 base pairs, by the year 2006 requires roughly 100 times beyond the total, current worldwide DNA sequencing capacity (M. V. Olson, 1993, Proc. Natl. Acad. Sci. USA 90:4338). Existing DNA sequencing methods, for example, mass spectrometry (T. D. Wood et al., 1995, Proc. Natl. Acad. Sci. USA 92:11451), sequencing by hybridization (R. Drmanac et al., 1993, Science 260:1649), chromatography (C. G. Huber et al., 1993, Nucl. Acids Res. 21:1061), acoustophoresis (J. S. Heyman, U.S. Pat. No. 5,192,450), and electrophoresis, are generally inadequate to meet this sequencing goal.
The above methods have various drawbacks. Mass spectrometry requires an expensive mass spectrometer. Because of this cost, it is unlikely that this method will have widespread applicability. Sequencing by hybridization is still relatively new and untested. Liquid chromatography is capable of performing rapid separation of double-stranded DNA fragments, but is limited by poor resolution. The single-base resolution necessary for sequencing has only been demonstrated for fragments smaller than 150 base pairs. In acoustophoresis, acoustic waves push fragments through a liquid medium. This method is limited by the similarity in the acoustic properties of DNA fragments of similar lengths, preventing effective separation.
Electrophoresis remains the most common method by far for DNA sequencing. All conventional electrophoretic methods are generally similar (F. Sanger et al., 1977, Proc. Natl. Acad. Sci. USA 74:5463; L. M. Smith, 1993, Science 262:530). A DNA sample is generally first amplified, that is the DNA chains are made to replicate, usually by the polymerase chain reaction ("PCR"). Next, from the amplified sample, chain terminating DNA polymerase reactions (first described by Sanger et al.) produce nested sets of DNA fragments labeled with one of four unique fluorescent dyes conjugated with one of the four chain terminating bases (either ddATP, ddCTP, ddGTP, or ddTTP). In a related method, the chains are cleaved by chemical means to produce a similar set of labeled fragments (M. Maxam et al., 1977, Proc. Natl. Acad. Sci. USA 74:560). These fragments are then separated according to their molecular size by a variety of electrophoretic techniques, and the unique dye labeling each chain terminating base is detected by its fluorescence. The DNA base sequence is reconstructed from the detected pattern of chain fragments.
The accuracy required in DNA fragment size determination depends on the application. For example, DNA sequencing reactions produce a mixture, called a "ladder," of fragments with lengths separated by single bases and require exact length determination. Other applications produce greater differences between the fragment lengths, and methods that provide rapid sizing, but not necessarily exact length information, are valuable. Typical of such applications are the generation of patterns of restriction fragment length polymorphism ("RFLP"), genotyping, linkage analysis, microsatellite analysis and other fragment analysis application.
In an electrophoretic separation, the DNA molecules are separated according to their rates of migration in an electric field. The electric driving force is proportional to the net charge of the molecule. For a uniformly charged biopolymer such as DNA, the driving force is proportional to the number of base pairs in the DNA fragment. Since in a material obeying Stokes' Law, such as a liquid, the friction coefficient is also proportional to the number of base pairs, the DNA fragments have electrophoretic drift velocities that are nearly identical and independent of fragment length. This means electrophoretic separation of DNA fragments is difficult in liquids or other media obeying Stokes' Law.
Therefore, instead of liquid media, cross-linked gels and uncross-linked polymer solutions are universally used in electrophoretic DNA separations. In these media, DNA does not obey Stokes' law, since the electrophoretic drift velocity decreases with increasing length or molecular weight. Thus, electrophoretic separation of biopolymers is ordinarily performed in a polymeric gel, such as agarose or polyacrylamide, in which separation of biopolymers with similar electric charge densities, such as DNA or RNA, depends on molecular weight. The non-Stokes' law dependence of the friction coefficient on the fragment size in a gel permits electrophoretic separation of DNA fragments of different lengths. Biopolymer fragments, therefore, exit the device in size order from small to large.
In a prevalent configuration, the electrophoretic gel is disposed as a thin sheet between two flat, parallel, rectangular glass plates. An electric field is established along the long axis of the rectangular configuration, and molecular migration is arranged to occur simultaneously in several paths, or "lanes," parallel to the electric field. To ensure high separation resolution, it is advantageous that gel throughout a migration lane be as uniform as possible (or homogeneous like a liquid) and for the lanes to be sufficiently separated to be clearly distinguishable.
It has proven difficult to make, or "to cast," uniform gels with uniform transport properties. One major problem is uneven gel shrinkage due to cross-linking during gel polymerization. The problems in casting a uniform gel also lead to difficulties in producing a uniform and reproducible loading region, into which sample mixtures are placed prior to separation. It is generally accepted that a separation medium with more reproducible transport properties (i.e., more like a homogeneous liquid) would have great utility.
In addition to high separation resolution, demands for more rapid electrophoresis have created additional problems for gel manipulation. Rapid electrophoresis is desirable for rapid, high capacity biopolymer analysis. This requires, primarily, stronger electric fields that exert greater forces on migrating molecules in order to move them at greater velocities. However, higher fields, voltages, and velocities lead to increased resistive heating in the gel, and consequently, significant thermal gradients in the gel. Such thermal gradients cause additional gel non-uniformities that further impair separation resolution.
To maintain resolution at higher voltages, ever smaller gel geometries are used so that damaging heat may be more readily conducted away. Thus, electrophoresis has been described in geometries where the parallel glass plates are spaced from 25 to 150 .mu.m apart, instead of the usual spacings which are typically greater than 1000 .mu.m (A. J. Kostichka et al., 1992, Bio/Technology 10:78). It has proven even more difficult to cast uniform gels of such thinness and to assure long, parallel, narrow, and closely spaced migration lanes in so thin a sheet.
In turn, to overcome these difficulties in thin gels, physical separation means have been used to keep lanes distinct. These separation means create yet a further set of problems. In one such method for producing physically distinct lanes, arrays of capillary tubes with diameters down to 100 .mu.m have been used (X. C. Huang et al., 1992, Anal. Chem. 64:2149). These capillary arrays are difficult to cast with uniform gels and difficult to load with samples of fragments. Easy loading is advantageous to minimize the time and cost of the separation setup, which is often labor-intensive. An alternative is to use a dilute polymer solution instead of a gel in each capillary (P. D. Grossman, U.S. Pat. No. 5,374,527). However, single base resolution in such solutions has been limited to DNA chains with fewer than 200 bases and loading the capillaries with samples remains difficult (A. E. Barron et al., 1993, J. Chromatogr. A 652:3; A. E. Barron et al., 1994, Electrophoresis 15:597; and Y. Kim et al., 1994, Anal. Chem. 66:1168). Other alternatives include producing physically distinct lanes by microfabrication of channels in an electrophoretic device (D. J. Harrison et al., 1992, Anal. Chem. 64:1926 and D. J. Harrison et al., 1993, Science 261:895). Electrodes can be deposited to provide precise control of the electrophoretic field (G. T. A. Kovacs et al., 1990, European Patent 0 376 611 A3 and D. S. Soane et al., U.S. Pat. No. 5,126,022). In another alternative to migration through gels, optical microlithography has been used to fabricate a quasi-two-dimensional array of migration obstacles for the electrophoretic separation of DNA (W. D. Volkmuth et al., 1992, Nature 358:600).
Small lane size coupled with the desirability of separating many samples in many migration lanes at once creates conflicting physical requirements. Simultaneous detection of fragments migrating in multiple lanes requires a spatially compact disposition of the migration lanes in order that all the lanes can be observed at once by a spectrograph of limited aperture. However, loading samples into migration lanes prior to separation requires physical access to the migration lanes that is easier and more rapid for widely spaced lanes. Conventional, flat-plate techniques have only straight, parallel lanes and cannot accommodate these divergent requirements.
Such problems with prior gel-based electrophoretic separation methods have motivated a search for new separation methods. A non-electrophoretic method for separation of particles that are electrically polarizable, but not charged, is based on differences in diffusivities in liquid of the particles. Only mega-base size DNA fragments have sufficient polarizability to be separated by this method (A. Ajdari et al., 1991, Proc. Natl. Acad. Sci. USA 88:4468; J. Rousselet et al., 1994, Nature 370:446; and J. F. Chauwin et al, 1994, Europhys. Lett. 27:421). This method uses an electric field that is periodic but asymmetric in space, substantially transverse to the direction of separation, and cycles temporally from on to off. When the asymmetric field is turned on, it attracts and traps polarizable particles into a series of spatially periodic attractive regions according to the known laws of electrostatics. When the potential is turned off, however, the particles are free to diffuse. Since smaller particles diffuse more rapidly, the cycling electric field causes a size separation of polarizable particles.
The polarization-based device is suited for separating particles on the order of the size of viruses, and may also be able to effect the separation of mega-base fragments of DNA, such as entire chromosomes (J. Rousselet et al., 1994, Nature 370:446). This particle size limitation is due to the requirement that the particles to be separated have polarizabilities sufficiently large to be attracted by fields that can be realistically created in a liquid. Since the attractive force varies as the square of the electric field, high voltages are needed. Separation of DNA fragments of a few 100's of bases in length, the sizes commonly produced by sequencing reactions or by RFLPs, is out of reach of this or similar polarization-based devices due to practical limits on electric field strength and voltages.
All the foregoing technical problems have hindered creation of a machine for rapid, concurrent analysis of large numbers of biopolymer samples at low cost and with minimal human intervention. The need for such a machine is widely felt in many areas of biology such as, for example, biological research, the Human Genome Project, the biotechnology industry, and clinical diagnosis.
Citation of references hereinabove shall not be construed as an admission that such reference is prior art to the present invention.